The invention discloses a high-yield mixing culturing method of phaeodactylum tricornutum bohlin and nitzschia closterium, aiming at solving the problems that the phaeodactylum tricornutum bohlin and the nitzschia closterium are individually cultured with an f/2 culture medium in an open cement pit, which causes low growth rate, low yield, easy polluting, and complex operation in the traditional culture method. The culture medium, disclosed by the invention, is characterized in that a mineral water barrel is utilized for mixing culturing of the phaeodactylum tricornutum bohlin and the nitzschia closterium, thus high fatty acid content can be obtained, the cost is low, the pollution is difficult to cause, the operation is easy, and the workload is greatly decreased; in addition, peptone, agar, maltose, potassium sorbate, glucose, sea mud extracting liquor, soil extract liquor, human urine, and cow and sheep manure leaching liquor are added to the culture medium, so that the nutrition is comprehensive and balanced, the growth speed of algae can be greatly raised, and the output is increased by 180%; moreover, the mixing culturing enables the mutualism of the two kinds of algae, and the two kinds of algae can absorb advantages mutually so as to promote each other; nutritive salt is fully utilized, so that the economic benefit is also increased.